Altered cytoplasmic-to-nuclear ratio of survivin is a prognostic indicator in breast cancer

Abstract

Purpose: Survivin (BIRC5) is a promising tumor biomarker. Conflicting data exist on its prognostic effect in breast cancer. These data may at least be partly due to the manual interpretation of immunohistochemical staining, especially as survivin can be located in both the nucleus and cytoplasm. Quantitative determination of survivin expression using image analysis offers the opportunity to develop alternative scoring models for survivin immunohistochemistry. Here, we present such a model.

Experimental design: A breast cancer tissue microarray containing 102 tumors was stained with an anti-survivin antibody. Whole-slide scanning was used to capture high-resolution images. These images were analyzed using automated algorithms to quantify the staining.

Results: Increased nuclear, but not cytoplasmic, survivin was associated with a reduced overall survival (OS; P = 0.038) and disease-specific survival (P = 0.0015). A high cytoplasmic-to-nuclear ratio (CNR) of survivin was associated with improved OS (P = 0.005) and disease-specific survival (P = 0.05). Multivariate analysis revealed that the survivin CNR was an independent predictor of OS (hazard ratio, 0.09; 95% confidence interval, 0.01-0.76; P = 0.027). A survivin CNR of >5 correlated positively with estrogen receptor (P = 0.019) and progesterone receptor (P = 0.033) levels, whereas it was negatively associated with Ki-67 expression (P = 0.04), p53 status (P = 0.005), and c-myc amplification (P = 0.016).

Conclusion: Different prognostic information is supplied by nuclear and cytoplasmic survivin in breast cancer. Nuclear survivin is a poor prognostic marker in breast cancer. Moreover, CNR of survivin, as determined by image analysis, is an independent prognostic factor.

Fig. 1.

Western blotting and immunohistochemical analysis using anti-survivin antibody.Western blot of survivin in a panel of breast cancer cell lines showing the specificity of the antibody. A, β-actin was used as a loading control. Survivin immunohistochemistry and H&E-stained cores. B, cytoplasmic survivin was scored manually.

Fig. 2.

Automated analysis of survivin immunohistochemistry reveals a relationship between nuclear survivin and poor prognosis. Immunohistochemistry of survivin with corresponding markup images of nuclear and cytoplasmic algorithms. High-power image showing nuclear and cytoplasmic algorithm markup images. A, note that nuclei are red in nuclear markup but blue in the cytoplasmic markup. The algorithm outputs were nuclear and cytoplasmic intensity, which were of a linear nature (histograms of outputs are available; Supplementary Fig. S2). B, RFC of nuclear and cytoplasmic intensity revealing four distinct clusters. C, error bar showing mean survivin cytoplasmic intensity and nuclear intensity, based on random forest clusters, shows greater separation of nuclear intensity compared with cytoplasmic intensity. Kaplan-Meier estimate of BCSS comparing clusters 1 to 3 (n = 66) with cluster 4 (n = 30; D) and OS and BCSS based on survivin nuclear autoscore (SNAS; E and F).

Fig. 3.

Survivin CNR is a prognostic factor in breast cancer. A, tumor cores with markup images showing a low and high CNR. Note marked difference in number of red pixels (identifying nuclear survivin) in the CNR < 5 core compared with the CNR > 5 core, which shows minimal nuclear staining. B, RFC of survivin CNR reveals four clusters. Kaplan-Meier estimate of OS based on RFC of survivin CNR (C) and OS and BCSS using a CNR of5 as a cutoff (D and E).